Neuroactive steroids are effective modulators of gamma-aminobutyric acidA receptors (GABAaRs), augmenting the actions of GABA at low concentrations and directly activating chloride channels at higher concentrations. Other steroids, particularly those with charged substituents at the 3-position, are negative modulators of GABAARs and other potentiators or inhibitors of N-methyl-D-aspartate receptors (NMDARs). Present evidence indicates that GABAAR potentiation and NMDAR inhibition represent two key mechanisms in clinical anesthesia. Over the past funding period, we identified a novel steroid that both potentiates GABAARs and inhibits NMDARs. This agent represent a potential new of anesthetics that combines two of the major features of presently used anesthetics in a single agent. In this present proposal, we will extend our studies of neuroactive steroids by addressing three specific aims: 1. To gain new information about structural requirements for steroid effects on GABAARs in cultured rat hippocampal neurons and Xenopus oocytes expressing defined GABAARs. These studies will include efforts to develop specific antagonists for steroid sites on GABAARs. 2. To understand mechanisms involved in neurosteroid-mediated modulation of hippocampal GABAergic synaptic transmission. Using a combination of synaptic recordings and studies using rapid agonist applications on isolated membrane. Using a combination of synaptic recordings and studies using rapid agonist applications on isolated membrane patches, these studies will examine mechanisms involved in steroid modulation. 3. To examine mechanisms involved in modulation of NMDARs by neuroactive steroids in hippocampal neurons and Xenopus oocytes. These studies include a combination of structure-activity and mechanistic studies based upon our initial observations with the novel steroid that enhances GABAARs and inhibits NMDARs. These studies have the potential to provide new information about neurosteroid effects in the CNS and a better understanding of mechanisms involved in steroid-mediated anesthesia.